OBJECTIVE:
An experimental in vitro study was carried out to evaluate the influence of cortical
bone thickness on ultrasound propagation velocity.METHODS: Sixty bone
plates were used, made from bovine femurs, with thickness ranging from 1 to 6
mm (10 of each). The ultrasound velocity measurements were performed using a device
specially designed for this purpose, in an underwater acoustic tank and with direct
contact using contact gel. The transducers were positioned in two ways: on opposite
sides, with the bone between them, for the transverse measurement; and parallel
to each other, on the same side of the bone plates, for the axial measurements.RESULTS: In the axial transmission mode, the ultrasound velocity speed
increased with cortical bone thickness, regardless of the distance between the
transducers, up to a thickness of 5 mm, then remained constant thereafter. There
were no changes in velocity when the transverse measures were made.CONCLUSION:
Ultrasound velocity increased with cortical bone thickness in the axial transmission
mode, until the thickness surpasses the wavelength, after which point it remained
constant. Level of Evidence: Experimental Study.

Keywords:
Ultrasonics. Acoustics. Bone and bones.

INTRODUCTION

The
use of ultrasound as a medical diagnostics method has generated considerable interest
due to the low cost, portability, ease of handling, possibility of managing to
generate images in real time, providing information on the physical properties
of tissues, non-invasiveness, and above all, having the fact that it does not
produce ionizing radiation as a characteristic.1

In
the last two decades the use of ultrasound for evaluation of bone quality through
the calculation of its propagation velocity was the subject of countless investigations,
emerging as an accurate and reproducible method, which can be used as an auxiliary
technique with bone densitometry in the assessment of osteoporosis and of the
clinical follow-up of patients. Studies on the normal bone consolidation process,
its disorders and the influence of a wide variety of types of implants when fractures
are treated surgically have been started recently, using the same methodology.
The results show that the technique is practicable, yet many aspects still need
to be studied for quantitative bone ultrasonometry to be validated as an auxiliary
method in connection with radiography and computed tomography in the evaluation
and follow-up of fractures.

The
objective of this study was to evaluate the influence of cortical bone thickness
on ultrasound propagation velocity, employing bovine femoral bone plates as an
experimental model and performing the quantitative ultrasonometry by the underwater
and direct contact technique.

MATERIAL
AND METHOD

Bovine
femur was the bone chosen to carry out the study because of the availability of
fresh and frozen pieces at cold-storage plants; due to the fact that they came
from animals with known weight, age and sex; as they had been sacrificed for consumption,
which would avoid the use of animals exclusively for the survey, and as this bone
presents thick cortex as a characteristic, making the idea of the study practicable.
We used 60 femurs (39 left and 21 right), from Nelore cattle, all males, aged
approximately three years and with 500 kg of weight.

The
femurs were then submitted to removal of all the soft parts, still frozen, and
only their diaphysis was used to make the bone plates. Each diaphysis enabled
the creation of just one plate, since the anterior surface of the bone was always
used as a means of standardizing the samples. The plates were made in a length
of 130mm, with a width of 30mm and different thicknesses, ranging from 1mm to
6mm. The minimum thickness of 1mm was owing to the fact that thinner plates were
hard to make and became excessively fragile; when over 6mm, it was no longer possible
to achieve uniformity of thickness throughout the length of the sample.

Once
they were ready, the bone plates were packed in properly identified individual
plastic bags, sealed and frozen at an average temperature of -20°C.

The
sample size was calculated using R software in version 2.6.2, assuming that six
different thicknesses would be compared in relation to sound velocity. Thus we
opted to work with 10 samples of each thickness and the ultrasonometric measurements
were taken under the following conditions: AUW (axial underwater) with a distance
of 3, 5 and 7cm between the transducers; TUW (transverse underwater); ADC (axial
direct contact) with a distance of 3, 5 and 7cm between the transducers and TDC
(transverse direct contact).

The
underwater sound velocity measurements were performed in an acrylic acoustic tank,
with a length of 36cm, height of 10cm and width of 7cm, dimensions considered
appropriate to adapt from small bone segments up to some whole bones. A circular
window was made in the geometric center of each side wall of the tank, for coupling
of the ultrasonic transducers precisely aligned with one another by their axial
axis.

Two
disc-shaped transducers (one emitter and one receptor) made from PZT-5 wafers,
a ceramic with piezoelectric properties, with a diameter of 25mm, were used for
the performance of the procedures. The transducers were connected to ultrasound
pulse generator-receptor-amplifier (Biotecnosis do Brasil®) equipment,
connected to an oscilloscope (Digital Storage Oscilloscope 3062A, Agilent Technologies®)
for visualization of the signal received. This equipment, in turn, is connected
to a microcomputer fed with a program for signal processing and for ultrasound
velocity calculation. The ultrasonic equipment used functions with a circuit that
generates narrow pulses with a frequency of 1 MHz. The input voltage in the source
transformer is adjustable, but it was set at 100 V, thus fixing the voltage applied
in the emitter transducer, with sufficient power for the pulse to cross the bone
sample without being totally attenuated.

The
signal received by the receptor transducer is amplified by a specific circuit,
featuring a selector switch that allows users to amplify the signal or not, with
3X amplification having been established for better visualization of the waves.
The oscilloscope visualizes wave reception and the microcomputer processes the
signs received and stores the information.

In
velocity calculation it is important to identify the point of arrival of the first
wave (FAS, or first arrived signal), which will define the travel time on the
path. (Figure 1) Several frames of references can be used to
confirm signal arrival. In this case it was defined as wave deflection greater
than 5% from the baseline, and that is calculated automatically by the computer
program.

The
equipment was calibrated using a polytetrafluorethylene cylinder, with known and
constant ultrasound propagation velocity. The cylinder was positioned between
the transducers so that the ultrasonic wave could be incident on the flat surface
of the piece. The room temperature was kept at 23°C. The ultrasound propagation
velocity was only measured in the water, and afterwards, with the polytetrafluorethylene
cylinder positioned inside it. In the case of the direct contact technique, contact
gel was used between the transducers and the polytetrafluorethylene piece. This
procedure was repeated before the evaluation of each bone plate, to ensure the
reproducibility of the measurements. The ultrasound propagation velocity in the
water and in the Teflon cylinder averaged 1,470 m/s and 1,156 m/s, respectively.

Before
the performance of the ultrasonic measurements, the bone plates were transferred
from the storage freezer to a domestic freezer, remaining at -12º C for 12
hours. After that, they were transferred to a refrigerator for a further 12 hours
at an average temperature of +4º C. Before the measurements, the assembled
pieces remained at a controlled room temperature of 23º C, the same as the
water in the acoustic tank.

The
measurements by direct contact between the transducers and the bone plates were
performed with the help of contact gel. Two types of assemblies were created,
one for measuring the ultrasound velocity through axial transmission, and the
other for transverse transmission.

For
the measurement by axial direct contact (ADC) we used rubber mounts that allowed
the exposure of the entire surface of the bone plate and the distance between
the transducers could be freely altered. (Figure 2) For the
measurement by transverse direct contact (TDC) a rubber mount was also used just
to support the assembly. (Figure 3)

For
the measurements by ADC, each group of bone plates with a particular thickness
was tested placing the transducers (using the center of the contact surface of
the transducer as a reference) at a distance of 3cm, 5cm and 7cm from one another,
gauged by means of prebuilt rubber molds, with measurements corresponding to each
distance, coupled to the transducers. In this manner we were able to evaluate
the influence of the cortical thickness and of the distance between the transducers
on the US propagation velocity. In the case of the measurements by TDC, the only
variable to be analyzed was the cortical thickness.

The
underwater measurements were performed with the help of the acoustic tank. Two
types of assembly were made, one to gauge the ultrasound velocity by axial transmission,
and the other, for transverse transmission. The same rubber mounts used in the
ADC technique were applied for the axial underwater (AUW measurement), allowing
the exposure of the entire surface of the bone plate with free alteration of the
distance between the transducers. In this situation, the windows of the side walls
of the tank were sealed and the transducers were positioned on the surface of
the bone plate through the upper opening. For the transverse underwater (TUW)
measurement, the transducers were coupled on the side windows and the distance
between them was kept constant to allow the accurate calculation of the US velocity.
(Figure 4)

Six
groups of bone plates were prepared for the performance of the study, each one
with 10-plate samples of the same thickness for each group. Each group was submitted
to analysis of the sound propagation velocity by the two techniques (direct contact
and underwater), in two different ways, by axial transmission and by transverse
transmission. (Table 1)

In
each specific case we performed three sequential measurements of the SV and extracted
the mean of the values obtained for each bone plate. After this, we calculated
the mean value corresponding to each group, which was employed in the statistical
calculations.

The
results were compared to evaluate the influence of cortical thickness on axial
and transverse US propagation velocity, to compare the bone ultrasonometry methods
(direct contact and underwater) and to evaluate the influence of the distance
between the transducers on the SV in axial transmission.

The
linear regression model with mixed effects (random and fixed effects) was used
to achieve the objectives. Linear mixed-effects models are used in data analysis
in which the responses are grouped (measurements repeated for the same individual)
and the supposition of independence between observations in the same group is
not adequate.2 These models are based on the assumption that their
residues have normal distribution with mean 0 and variance Σ2.
In situations in which such an assumption was not observed, transformations in
the response variable were used. The Bonferroni simultaneous confidence interval
was used with consequent correction of p-value, in order to guarantee that the
simultaneous comparisons between the means maintain 95% of confidence.3
This procedure was executed through the SAS® 9.0 software (SAS
Institute Inc., SAS/STAT® User's Guide, Version 9.0, Cary, North
Carolina, USA), using PROC MIXED.

RESULTS

The
USPV increased consistently with the increase in the thickness of the bone plates,
in the axial direct contact (ADC) and underwater (AUW) measurements, but presenting
uniformity in the transverse direct contact (CDT) or underwater (TUW) measurements,
practically without variation accompanying the thickness of the plates. On the
other hand, the distance between the transducers (3, 5 and 7cm) in the axial director
contact (ADC) or underwater (AUW) measurements did not produce significant differences
in the velocities beyond those observed for the thickness of the plates.

In
the ADC measurements, the mean value of the USPV increased from 3491.40 m/s in
the thickness of 1mm to 4201.20 m/s in the thickness of 6mm, for the distance
of 3cm between the transducers. For the distance of 5cm, the mean USPV increased
from 3497.50 m/s in the thickness of 1mm to 4200.30 m/s in the thickness of 6mm.
For the distance of 7cm, the mean USPV increased from 3497.90 in the thickness
of 1mm to 4200.60 in the thickness of 6mm. (Table
2) The differences between the measurements were significant for all the comparisons
(p<0.0001), with the exception of those between the thicknesses of 5 and 6mm
(p=1), in the three different distances between the transducers. For each individual
thickness from 1 to 6 mm, there were no significant differences observed for any
comparison between the measurements in keeping with the distance between the transducers,
of 3, 5 and 7cm, evidencing that this parameter is not important, within the limits
investigated.

In
the AUW measurements, the mean USPV increased from 3498.90 m/s in the thickness
of 1mm to 4200.20 m/s in the thickness of 6mm, for the distance of 3cm between
the transducers. For the distance of 5cm, the mean USPV increased from 3493.10
m/s in the thickness of 1mm to 4201.10 m/s in the thickness of 6 mm, and for the
distance of 7cm, the mean USPV increased from 3491.70 m/s in the thickness of
1mm to 4200.10 m/s in the thickness of 6mm. (Table
3) The differences between the measurements were significant for all the comparisons
(p<0.0001), with the exception of that between the thicknesses of 5 and 6 mm
(p=1), in the three different distances between the transducers. For each individual
thickness from 1 to 6mm, there were no significant differences observed for any
comparison between the measurements in keeping with the distance between the transducers,
of 3, 5 and 7cm, once again demonstrating that this parameter is not important,
within the limits investigated.

Comparisons
were also made for the same thickness (from 1 to 6mm) of the bone plate and the
same distance (3, 5 and 7cm) between the transducers, analyzing the ADC and AUW
techniques, with no significant differences having been demonstrated for any comparison,
indicating that the two techniques are equivalent.

Grouping
the aforesaid data, knowing that there is no significant difference between the
velocities with the two techniques (direct contact and underwater), we can set
out the results of the axial measurements in a graph that allows us to visualize
the pattern of growing ultrasound propagation velocity with the increase in plate
thickness, until stabilization from five millimeters of thickness. (Figure
5) In the TDC and TUW measurements, the mean USPV varied very slightly between
the thicknesses of 1 to 6mm, remaining between the minimum of 3438.40 m/s and
the maximum of 3441.50 m/s for the first, and between the minimum of 3436.90 m/s
and the maximum of 3442.90 m/s for the second. (Table
4) There was no significant difference between the measurements of TDC (p=1)
and, also, of TUW (p=1), when comparing the different thicknesses.

Comparisons
were also made for the same thickness (from 1 to 6 mm) of the bone plate analyzing
the TDC and TUW techniques, without any significant differences having been demonstrated
for any comparison, which also indicates that the two techniques are equivalent.
(Figure 6)

DISCUSSION

The
quantitative evaluation of the quality of bone tissue by means of the measurement
of the ultrasound conduction speed has been the subject of countless investigations,
mainly geared towards the measurement of osteoporosis and of bone healing. With
regard to the measure of osteoporosis, the current literature is richer and provides
solid subsidies for clinical applicability. On the other hand, this is not true
when the focal point is the bone healing process. There are in vitro and in vivo
studies, with strong evidence that the method can be applied clinically, yet there
is still a lot to be understood until it can be standardized and the results considered
reliable.

The
fracture consolidation process in humans and animals is usually evaluated through
radiographs or computerized tomography, methods that involve the use of ionizing
radiation with known deleterious effects on the tissues.4 This, added
to the fact that the bone callus is only visible upon examination if sufficiently
calcified, that bone consolidation does not always involves callus formation,
as in cases of rigidly fixed diaphyseal fractures submitted to osteosynthesis
by the absolute stability method, and that the fracture line can often not be
visualized as there are overlapping metal implants, justifies the search for an
alternative resource in this field. In addition, there is the fact that it may
be necessary to obtain many and repeated radiographs over the course of the treatment,
exposing the patient to an overdose of radiation, with significant potential for
secondary lesions, particularly in children and pregnant women.

The
availability of a resource that involves the employment of a non-ionizing physical
agent and that can be used in the initial phases of consolidation would be very
useful, especially when many successive evaluations are necessary. Magnetic resonance
(MR) has all the above characteristics, but is costly, not always available and
the images obtained suffer the influence of metal implants, hindering an adequate
interpretation. The use of conventional ultrasound is a possibility, since the
method is inexpensive when compared to the other techniques, widely available
and easy to handle. However, the images obtained are frequently hard to interpret,
with limited reproducibility and dependent on the examiner's experience. The equipment
that quantitatively evaluates transosseous ultrasound conduction, such as that
used in the diagnosis of osteoporosis and osteopenia, has all the ideal characteristics
to be an auxiliary method in the study of the fracture consolidation process,
since this equipment has the abovementioned advantages of ultrasound, as well
as objective results.

The
use of ultrasonometry for bone evaluation was initially described by Siegel et
al.5 in 1958, using rabbit tibias. Its use for the evaluation of bone
density through BUA (Broadband Ultrasound Attenuation) was described by Langton
et al.6, and is capable of predicting the quality and the quantity
of bone mass, gaining space in recent years with several commercially available
models. The use of ultrasonometry for monitoring the fracture consolidation process
is more recent, and few studies demonstrate its clinical applicability;7,8
however, without adequate standardization, due mainly to methodological issues.

In
our area, Barbieri et al.9 conducted an in vitro study on the use of
transverse underwater ultrasonometry to evaluate the consolidation of transverse
diaphyseal osteotomies of sheep tibia in different periods, demonstrating that
the velocity of ultrasound propagation through the bone increases as the consolidation
process progresses. This investigation was carried out with a tibial osteotomy
external fixation model, which favors bone callus consolidation, a process entirely
different from the direct consolidation obtained with the rigid fixation plates
using axial compression. This latter type of fixation has become increasingly
frequent in the clinical practice, equally entailing an increase in complications
such as the delay of consolidation, which can have its diagnosis hindered by the
overlapping of the implant as presented previously. Accordingly, Bezuti10
proposed the in vitro study of the interaction between bone and metal fracture
fixation plate, by the measurement of the ultrasound propagation velocity in different
planes, showing that the method is efficient in detecting mid-diaphyseal transverse
osteotomy furrow of sheep tibia, with no significant influence of the implant
on dependency of the plane of incidence of the ultrasonic waves.

Bone
ultrasonometry can be performed with the transducers positioned in two different
ways; on opposite sides, with the bone between them, for the transverse measurement;
or parallel to each other, on the same cortical surface, for the axial measurement.
The technique used can also differ between that by direct contact, in which the
transducers are placed directly on the bone surface with the help of a contact
gel, or the underwater technique, in which the bone is completely submerged in
a tank of water. The aquatic medium presents better conditions for the propagation
of sound and the study of its velocity, yet the direct contact technique is interesting
as the in vivo underwater analysis is often unfeasible.

Two
were questions that motivated this study. The first is regarding the type of influence
that the thickness of the cortical bone would have on ultrasound propagation velocity,
since we know that the thickness can be different according to sex, age, race,
level of physical activity and presence of metabolic diseases, with possible impacts
on the ultrasonic evaluation. The second concerns the degree of equivalence existing
between ultrasonometric techniques, in this case the underwater technique, and
the direct contact technique. An in vitro study was idealized for this purpose,
using homogeneous bone plates, with all the possible variables well controlled.
This kind of comparison is unviable in whole bones, which present an unfavorable
relief and significant variability between one another.

The
experimental model chosen was that of using bone plates made from the anterior
diaphyseal cortex of bovine femur. Bovine femur was chosen for the ease in acquiring
samples of fresh and frozen pieces at cold-storage plants, from animals with known
weight, age and sex; as it was not necessary to sacrificed animals for the survey,
and as this bone presents thick cortex as a characteristic, making the idea of
the study viable. The anterior surface of the bone was standardized to make the
plates. Due to the surface anatomy of this bone and its average diameter, the
largest plate that can be manufactured and always replicated, was 130mm long by
30mm wide, dimensions sufficient for adequate adaptation to the ultrasonic transducers,
in a diameter of 25mm. As regards thickness, it was not always possible to obtain
plates thicker than 6mm and that were regular all along their length. Likewise,
plates with a thickness of less than 1mm were hard to produce and became very
fragile. Therefore, the plates from the study ranged from 1mm to 6mm in thickness.
To evaluate larger thicknesses, the only possible option would be the use of an
animal of greater size or the use of synthetic bone models.

The
equipment used for the measurements was built by a specialized company, for the
specific purposes of bone ultrasonometry, and could be adapted both for the underwater
and for the direct contact measurements, having already been used in previous
investigations. It is a prototype that should be implemented for commercial availability
and is endowed with digital technology. The computer program developed allows
users to measure the ultrasound velocity in both situations (underwater and by
direct contact) with high reliability.

As
specified by Hill11 the main parameter chosen for analysis was ultrasound
propagation velocity through the bone, as it is considered the essential property
of acoustic propagation in tissues. Pocock et al.12 showed that ultrasound
propagation velocity varies with the temperature of the medium of reference (water)
and of the actual sample to be analyzed, which is the reason why the temperature
of the water and of all the samples was also standardized during the execution
of the analyses. The actual velocity is calculated by means of an equation that
can vary according to the source consulted, and in this case, we used that proposed
by Evans and Tavakoli.13

Sievänen
et al.14 commented on the need for at least three ultrasound propagation
velocity measurements for each region of interest, which would enhance the reliability
of the results obtained. This guideline was followed in the present study, and
after the measurements we calculated the mean to arrive at the final value.

The
ultrasound propagation velocity results obtained showed that the distance between
the emitter and receptor transducers in axial transmission did not influence the
measurements, regardless of the technique used, whether by direct contact or underwater.
Three distances were used between the transducers in this study: 3cm, 5cm and
7cm. Longer distances were not practicable due to the power of the signal generating
equipment and to the high impedance of the bone.

There
was no difference in velocity, either, when comparing the different thicknesses
of the bone plates with transverse transmission, regardless of the technique used,
whether via direct contact or underwater. This data is theoretically expected,
since the thicker the plates, the greater the distances and the longer the time
needed to cover them.

Another
important fact is that the values obtained for ultrasound velocity in bovine cortical
bone was similar to the values reported previously by Evans and Tavakoli,13
which validates the techniques used in this study and the device developed for
this purpose.

It
is possible to notice that all the measurements performed, with axial or transverse
transmission, failed to present differences in keeping with the technique used.
Although not the initial objective of the study, we ended up demonstrating that
the direct contact technique is comparable with the underground technique. This
comparison was only possible due to the object studied, in this case relatively
regular bone plates without significant differences between each other. This would
not be possible if whole bones had been used, as these differ from one another,
in spite of being from the same species and from the same limb. Moreover, superficial
anatomical irregularities, even though small, mean that the coupling of the transducers
is not complete and this can contribute to the difference in results when compared
with the underwater technique. Until now, this technique was considered more reproducible
since water is an excellent conductor of sound waves. However, the direct contact
technique appears to be more adaptable to clinical situations, as it is possible
to collimate the area of interest to be analyzed with greater ease than in an
acoustic tank.

As
already demonstrated previously by Njeh et al.15 sound velocity in
axial transmission depends on the thickness of the cortical bone; the thicker
the bone, the higher the velocity. This fact is true up to a certain limit, and
the authors postulate that velocity increases up to the point where the wavelength
is even smaller than the thickness of the cortex. The same fact was observed in
this study, in which the ultrasound propagation speed increased progressively
from about 3,500m/s in the bone plates with a thickness of 1mm (in both techniques,
by direct contact and underwater), passing through 3,650m/s in the 2mm plates,
3,800m/s with 3mm, 4,000m/s with 4mm, to arrive at 4,200m/s with 5mm. The velocity
remained constant at 4,200m/s in the thickness of 6mm. If we take into account
the fact that the frequency of the device is 1 MHz and that the wavelength is
calculated by dividing velocity by frequency, we obtain approximately 4.3mm as
the wavelength value, since it was the maximum velocity reached in the samples.
Therefore, the velocity is not expected to change any more, even if the sample
thickness increases, since the thickness has already exceeded the wavelength starting
from 5mm.

For
the plates of lesser thickness (1 to 4mm), the ultrasonic wavelength was always
equal to or greater than the thickness. In these situations, wave conduction occurs
throughout the bone thickness and not only on its surface, so that it reflects
the physical properties of the bone with greater precision. When the wavelength
is smaller than the thickness, its conduction changes, becoming superficial and
more rapid, but not reflecting the integral properties of the bone.16,17

Knowing
the ratio between cortical bone thickness and ultrasound propagation velocity
is extremely important for the continuity of studies and the standardization of
bone ultrasonometry. Age and osteometabolic diseases alter the thickness of the
bone cortex and influence the results obtained, besides the actual mean variability
of cortical thickness of the bones of the human body. We know that the mean cortical
thickness, measured by radiographic examination, is 1.7mm for the proximal phalanx
of the fingers of the hand, 2 to 3mm for the metacarpals, 3 to 3.6mm for the proximal
region of the radius, 5 to 8 mm for the tibial diaphysis and 2.3 to 7.4mm for
the femoral diaphysis.18 Accordingly, with the methodology employed
in this study, the maximum wavelength was 4.2mm, in the thicknesses of 5 and 6mm.
Nevertheless, there are many situations in which ultrasound velocity can be directly
affected by the thickness of the bone cortex above the studied limits, which opens
up a perspective for new investigations.

The
continuity of research in quantitative bone ultrasonometry should focus on applicability
in real clinical situations, as well as the influence of implants, that of the
geometry of the different patterns of fractures and of their consolidation, and
of the physical conditions of bones. Considering the envelope of soft parts around
the bones and the difficulties involved in using the underwater technique, the
technique of direct contact with the help of contact gel appears, up to this point,
to be the most likely path for bone ultrasonometry to occupy its space as a safe,
low-cost auxiliary method free of ionizing radiation in the evaluation of the
bone healing process, irrespective of the therapeutic approached used, whether
conservative or surgical.

CONCLUSION

The
thickness of the cortical bone influences ultrasound propagation velocity, when
the axial transmission technique is employed. The greater the thickness, the higher
the speed, up to the point where the thickness exceeds the wavelength. From this
point on, the velocity remains constant. Both bone ultrasonometry techniques,
via direct contact and underwater, proved practicable and had comparable results.

Study
conducted at the Department of Biomedicine, Medicine and Rehabilitation of the
Musculoskeletal System. of Faculdade de Medicina de Ribeirão Preto da Universidade
de São Paulo - Ribeirão Preto, SP, Brazil. All the authors declare
that there is no potential conflict of interest referring to this article.